The subject of the present invention is a DNA sequence encoding thanatin, a vector containing it for the transformation of a host organism, and the method of transforming the said organism.
The invention relates more particularly to the transformation of plant cells and plants, the thanatin produced by the transformed plants conferring on them resistance to diseases, in particular of fungal origin.
An increasing need already exists for making plants resistant against diseases, in particular fungal diseases, in order to reduce, or even eliminate, the need for treatment with antifungal protection products, with a view to protecting the environment. One means of increasing this resistance to diseases consists in transforming the plants so that they produce substances capable of providing their defence against these diseases.
Various substances of natural origin, in particular peptides, are known which exhibit bactericidal or fungicidal properties, in particular against the fungi responsible for plant diseases. However, the problem consists in finding such substances which not only can be produced by transformed plants, but can still preserve their bactericidal or fungicidal properties and confer them on the said plants. For the purposes of the present invention, bactericidal or fungicidal is understood to mean the actual bactericidal or fungicidal properties and the bacteriostatic and fungistatic properties.
Thanatin is a peptide produced by bacterial induction on adult Psodius sp, preferably maculiventris. Its preparation by bacterial induction is described in patent application FR 2,733,237, as well as its antifungal and antibacterial properties in vitro.
After having first identified the thanatin gene, it was also found that it could be inserted into a host organism, in particular a plant, in order to express the thanatin and confer on the said host organism properties of resistance to fungal diseases and to diseases of bacterial origin, providing a particularly advantageous solution to the problem stated above.
The subject of the invention is therefore first a nucleic acid fragment encoding thanatin, a chimeric gene comprising the said fragment encoding thanatin as well as heterologous regulatory elements at the 5xe2x80x2 and 3xe2x80x2 positions which can function in a host organism, in particular in plants, and a vector for transforming the host organisms containing this chimeric gene, and the transformed host organism. It also relates to a transformed plant cell containing at least one nucleic acid fragment encoding thanatin and a disease-resistant plant containing the said cell, in particular regenerated from this cell. It finally relates to a method of transforming plants to make them resistant to diseases, in which method a gene encoding thanatin is inserted by means of an appropriate vector.
Thanatin is understood to mean according to the invention any peptide comprising essentially the peptide sequence of 11 amino acids which is described in patent application FR 2,733,237, as well as the equivalent homologous sequences in which certain amino acids are replaced by different but equivalent amino acids at sites which do not induce substantial modification of the antifungal or antibacterial activity of the said homologous sequence. Peptide sequence comprising essentially the peptide sequence described in patent application FR 2,733,237 is understood to mean not only the sequence defined by the sequence identifier No. 1 (SEQ ID NO 1), but also such a sequence comprising at either of its ends, or at both, peptide residues necessary for its expression and targeting in a host organism, in particular a plant cell or a plant.
Thanatin is a peptide of formula (I)(SEQ ID NO:14):
Xaa-Ile Ile Tyr Cys Asn Arg Arg Thr Gly Lys Cys-Xabxe2x80x83xe2x80x83(I)
in which:
Xaa is NH2 or a variable residue having a sequence comprising from 1 to 10 amino acids, and
Xab is OH or a variable residue having a sequence comprising from 0 to 5 amino acids.
Advantageously, when Xaa comprises at least one amino acid, the latter is one of the 20 base amino acids and more particularly chosen from the group comprising Gly, Ser, Lys, Pro and Val. When Xab comprises at least one amino acid, the latter is one of the 20 base amino acids and more particularly chosen from the group comprising Gln, Arg and Met.
According to a preferred embodiment of the invention, the two cysteine residues of the peptide of formula (I) form an intramolecular disulphide bridge.
The present invention therefore relates first to a nucleic acid, in particular a DNA, fragment encoding the thanatin defined above. It may be, according to the invention, a fragment isolated from Psodius sp, preferably maculiventris, or alternatively a derived fragment, suitable for the expression of thanatin in the host organism where the peptide will be expressed. The nucleic acid fragment may be obtained using standard methods of isolation and purification, or alternatively by synthesis according to the customary techniques of successive hybridizations of synthetic oligonucleotides. These techniques are in particular described by Ausubel et al.
According to the present invention, xe2x80x9cnucleic acid fragmentxe2x80x9d is understood to mean a nucleotide sequence which may be of the DNA or RNA type, preferably of the DNA, in particular cDNA, especially double-stranded, type.
According to one embodiment of the invention, the nucleic acid fragment encoding thanatin comprises the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO 1), a homologous sequence or a sequence complementary to the said sequence.
Advantageously, the nucleic acid fragment according to the invention comprises the DNA sequence described by the sequence identifier No. 2 (SEQ ID NO 2), a homologous sequence or a sequence complementary to the said sequence.
xe2x80x9cHomologousxe2x80x9d is understood to mean according to the invention a nucleic acid fragment having one or more sequence modifications relative to the nucleotide sequence described by the sequence identifier No. 1 or No. 2 and encoding thanatin. These modifications may be obtained according to the customary mutation techniques, or alternatively by choosing the synthetic oligonucleotides used in the preparation of the said sequence by hybridization. Given the multiple combinations of nucleic acids which may lead to the expression of the same amino acid, the differences between the reference sequence described by the sequence identifier No. 1 or No. 2 and the homologue may be great, especially since a DNA fragment of less than 100 nucleic acids in size, which can be produced by synthesis, is involved. Advantageously, the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications are generally neutral, that is to say that they do not affect the primary sequence of the resulting thanatin.
The present invention also relates to a chimeric gene (or an expression cassette) comprising a coding sequence as well as heterologous regulatory elements at the 5xe2x80x2 and 3xe2x80x2 positions which can function in a host organism, in particular plant cells or plants, the coding sequence comprising at least one DNA fragment encoding thanatin as defined above.
Host organism is understood to mean any higher or lower mono- or pluricellular organism into which the chimeric gene according to the invention may, be introduced, for the production of thanatin. It consists of in particular bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, or preferably plant cells and plants. xe2x80x9cPlant cellxe2x80x9d is understood to mean according to the invention any cell derived from a plant and which may constitute undifferentiated tissues such as calli, and differentiated tissues such as embryos, plant portions, plants or seeds.
xe2x80x9cPlantxe2x80x9d is understood to mean according to the invention any differentiated multicellular organism capable of photosynthesis, in particular monocotyledones or dicotyledones, more particularly cultivated plants intended or otherwise as animal feed or for human consumption, such as maize, wheat, colza, soya bean, rice, sugar cane, beet, tobacco, cotton and the like.
The regulatory elements necessary for the expression of the DNA fragment encoding thanatin are well known to persons skilled in the art depending on the host organism. They comprise in particular promoter sequences, transcription enhancers, transit peptides, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to persons skilled in the art.
The nucleic acid fragment according to the invention may also comprise a nucleic acid sequence fused in 5xe2x80x2 and/or 3xe2x80x2 to the sequence encoding thanatin, so as to obtain a xe2x80x9cprotein-thanatinxe2x80x9d fusion protein, whose cleavage by the enzymatic systems of the host organism allows the release of thanatin. This thanatin-fused protein may be a signal peptide or a transit peptide which makes it possible to control and orient the production of thanatin in a specific manner in a part of the host organism, such as for example the cytoplasm, the cell membrane, or in the case of plants in a particular type of tissue or in the extracellular matrix.
According to one embodiment, the transit peptide may be a signal for chloroplast or mitochondrial addressing, which transit peptide is then cleaved in the chloroplast or the mitochondria.
According to another embodiment of the invention, the signal peptide may be an N-terminal signal or xe2x80x9cprepeptidexe2x80x9d, optionally in combination with a signal responsible for retaining the protein in the endoplasmic reticulum, or a peptide for vacuolar addressing or xe2x80x9cpropeptidexe2x80x9d. The endoplasmic reticulum is the site where operations of maturation of the protein produced, such as for example the cleavage of the signal peptide, are carried out by the xe2x80x9ccellular machineryxe2x80x9d.
The invention relates more particularly to the transformation of plants. As regulatory promoter sequence in plants, there may be used any promoter sequence of a gene which is expressed naturally in plants, in particular a promoter of bacterial, viral or plant origin such as, for example, that of a gene for the small subunit of ribulose biscarboxylase (RuBisCO) or of a plant virus gene, for example that of cauliflower mosaic (CAMV 19S or 35S), or a promoter inducible by pathogens such as tobacco PR-1a or asparagus AoPRT-L, it being possible for any known suitable promoter to be used. Preferably, a regulatory promoter sequence is used which promotes the overexpression of the coding sequence constitutively or inducibly by a pathogen attack, such as for example that comprising at least one histone promoter as described in application EP 0,507,698.
According to the invention, it is also possible to use, in combination with the regulatory promoter sequence, other regulatory sequences which are situated between the promoter and the coding sequence, such as transcription enhancers such as for example the tobacco mosaic virus (TMV) translation enhancer described in application WO 87/07644, or the tobacco etch virus (TEV) translation enhancer described by Carrington and Freed, or transit peptides, either single or double, and in this case optionally separated by an intermediate sequence, that is to say comprising, in the direction of transcription, a sequence encoding a transit peptide of a plant gene encoding a plastid localization enzyme, a portion of sequence of the N-terminal mature portion of a plant gene encoding a plastid localization enzyme, and then a sequence encoding a second transit peptide of a plant gene encoding a plastid localization enzyme consisting of a portion of sequence of the N-terminal mature portion of a plant gene encoding a plastid localization enzyme, as described in application EP 0,508,909. As transit peptide, there may be mentioned the signal peptide of the tobacco PR-1a gene described by Cornelissen et al., represented with its coding sequence by the sequence identifier No 3.
The sequence encoding the fusion protein signal peptide PR-1a-thanatin and this fusion protein also form part of the present invention. This sequence is in particular described by the sequence identifier No. 5, more particularly the coding part of this sequence, corresponding to bases 12 to 164.
As regulatory terminator or polyadenylation sequence, there may be used any corresponding sequence of bacterial origin, such as for example the nos terminator from Agrobacterium tumefaciens, or alternatively of plant origin, such as for example a histone terminator as described in application EP 0,633,317.
According to the present invention, the chimeric gene may also comprise a selectable marker suitable for the transformed host organism. Such selectable markers are well known to persons skilled in the art. They may be a gene for resistance to antibiotics, such as penicillin, or alternatively a gene for tolerance of herbicides for plants.
The present invention also relates to a cloning or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above. This vector comprises, in addition to the above chimeric gene, at least one replication origin. This vector may consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the chimeric gene according to the invention. Such transformation vectors, depending on the host organism to be transformed, are well known to persons skilled in the art and are widely described in the literature.
For the transformation of plant cells or plants, they may consist in particular of a virus which may be used for the transformation of developed plants and containing, in addition, its own elements for replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.
The subject of the invention is also a method of transforming host organisms, in particular plant cells, by integration of at least one nucleic acid fragment or a chimeric gene as defined above, which transformation may be obtained by any known appropriate means widely described in the specialist literature and in particular the references cited in the present application, more particularly by the vector according to the invention.
A series of methods consists in bombarding cells or protoplasts with particles to which the DNA sequences are attached. Another series of methods consists in using, as means of transferring into the plant, a chimeric gene inserted into a Ti plasmid from Agrobacterium tumefaciens or an Ri plasmid from Agrobacterium rhizogenes. 
Other methods may be used, such as microinjection or electroporation or alternatively direct precipitation by means of PEG.
Persons skilled in the art will make the choice of the appropriate method depending on the nature of the host organism, in particular the plant cell or the plant.
The subject of the present invention is also the host organisms, in particular plant cells or plants, transformed and containing an effective quantity of a chimeric gene comprising a sequence encoding the thanatin defined above.
The subject of the present invention is also the plants containing transformed cells, in particular the plants regenerated from transformed cells. The regeneration is obtained by any appropriate method which depends on the nature of the species, as for example described in the references above.
For the methods of transforming plant cells and of regenerating plants, the following patents and patent applications may be mentioned: U.S. Pat. Nos. 4,459,355, 4,536,475, 5,464,763, 5,177,010, 5,187,073, EP 267,159, EP 604 662, EP 672 752, U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,371,014, 5,478,744, 5,179,022, 5,565,346, 5,484,956, 5,508,468, 5,538,877, 5,554,798, 5,489,520, 5,510,318, 5,204,253, 5,405,765, EP 442,174, EP 486,233, EP 486,234, EP 539,563, EP 674,725, WO 91/02071 and WO 95/06128.
The present invention also relates to the transformed plants derived from the cultivation and/or crossing of the above regenerated plants, as well as the seeds of transformed plants.
The plants thus transformed are resistant to certain diseases, in particular to certain fungal or bacterial diseases. Consequently, the DNA sequence encoding thanatin may be integrated with the main objective of producing plants resistant to the said diseases, thanatin being effective against fungal diseases such as those caused by Cercospora, in particular Cercospora beticola, Cladosporium, in particular Cladosporium herbarum, Fusarium, in particular Fusarium culmorum or Fusarium graminearum or by Phytophthora, in particular Phytophthora cinnamomi. 
The chimeric gene may also advantageously comprise at least one selectable marker, such as one or more herbicide tolerance genes.
The DNA sequence encoding thanatin may also be integrated as a selectable marker during the transformation of plants with other sequences encoding other peptides or proteins of interest such as, for example, herbicide tolerance genes.
Such herbicide tolerance genes are well known to a person skilled in the art and are in particular described in patent applications EP 115 673, WO 87/04181, EP 337 899, WO 96/38567 or WO 97/04103.
Of course, the transformed cells and plants according to the invention may comprise, in addiction to the sequence encoding thanatin, other heterologous sequences encoding other additional peptides capable of conferring on the plant resistance to other diseases of bacterial or fungal origin.
The other sequences may be integrated by means of the same vector comprising a chimeric gene, which comprises a first sequence encoding thanatin and at least one other sequence encoding another peptide or protein of interest.
They may also be integrated by means of another vector comprising at least the said other sequence, according to the customary techniques defined above.
The plants according to the invention may also be obtained by crossing parents, one carrying the gene according to the invention encoding thanatin, the other carrying a gene encoding at least one other peptide or protein of interest.
Among the sequences encoding other antifungal peptides, there may be mentioned that encoding drosomycin, which is described in patent application FR 2,725,992 and by Fehlbaum et al. (1994), and in the unpublished patent application FR 97 09115 filed on Jul. 24, 1997, or that encoding androctonin described in patent application FR 2,745,004 and in unpublished patent application FR 97 10362 filed on Aug. 20, 1997.